Most therapeutic proteins are glycoproteins where oligosaccharides are covalently bonded to amino acid residues as they pass through the secretory pathway. The sugar moieties are known to greatly affect biological activity and function of the glycoproteins. To date, to solve the problems such as immune response in the body, therapeutic glycoproteins are commonly produced using animal cell expression systems. However, there are drawbacks to animal cell culture systems, which include low yield, high cost, and potential viral and prion contamination. In this regard, many attempts have been made to use, as an alternative to animal cell expression systems, yeast expression systems, which are efficient and high-yield expression systems, and share the early steps of the N-linked glycosylation pathway of higher animal cells.
Microbial eukaryotes, yeasts have advantages of rapidly producing a high concentration of proteins, being easily genetically engineered, and having no risk of infections by human or animal virus and prion to ensure safety. However, the final oligosaccharides synthesized in yeasts have different type of sugar moieties from those of human, and thus may cause immune responses in animal cells. To solve the above problems, there is a need for glycotechnology, by which the yeast glycosylation pathway is remodeled to express glycoproteins having oligosaccharides similar to those of human glycoproteins.
A traditional yeast, Saccharomyces cerevisiae has a hypermannosylated N-linked oligosaccharide composed of a series of 50 to 200 mannose residues attached to a core oligosaccharide chain and decorated with the terminal alpha 1,3-linked mannoses (Dean, Biochim. Biophys. Acta., 1426, p.309-322, 1999). In addition, mannosylphosphate is added to the core and outer chain of oligosaccharide (Ballow, 1990, Methods Enzymol. 185: 440-470), and a glycoprotein with mannosylphosphorylated oligosaccharide was reported to induce immune responses when injected to animals (Rosenfeld and Ballou, 1974, J. Biol. Chem. 249: 2319-2321). Thus, there is an attempt to humanize glycosylation pathway by disrupting OCH1 and MNN4 genes, which mediates outer chain initiation and participates in addition of mannosylphosphate, respectively (Jigami and Odani, Biochim, Biophys. Acta 1426, 335-345, 1999). However, mannosylphosphorylation was not completely regulated in MNN4-disrupted strains, even though the extent was less than that in a wild-type strain (Odani et al., Glycobiology, 6, p.805-810, 1996).
There is an attempt to humanize the glycosylation pathway by eliminating mannosylphosphorylation in a methylotrophic yeast, Pichia pastoris, as well as in the traditional yeast. A PNO1 (Phosphorylmannosylation of N-linked Oligosacharides) gene, which plays an important role in mannosylphosphorylation in Pichia pastoris, was cloned by using a sequence of MNN4 gene of Saccharomyces cerevisiae as a probe (Miura et al., Japan), and there is a report that the mannosylphosphorylation can be controlled by the elimination of PNO1 gene (WO 01/88143; Miura et al., 2004, Gene 324; 129-137).
However, it was found by GlycolFi Inc. that the disruption of the PNO1 may suppress the mannosylphosphorylation, but does not completely eliminate it, leading to application of the invention (US2006/0160179). In the invention, a BLAST search was performed for the amino acid sequence of Mnn4 protein (SEQ ID NO: 19) from Saccharomyces cerevisiae against the genome of Pichia pastoris (Integrated Genomics, Chicago, Ill.). This search resulted in the identification of three genes, which were designated as MNN4A, MNN4B and MNN4C, respectively. They also found that the mannosylphosphorylation can be completely eliminated by double disruption of MNN4A and PNO1 genes.
A dimorphic, non-pathogenic yeast, Yarrowia lipolytica has been used on a large scale for the production of citric acid and of single-cell proteins, and is characterized by excessive secretion of extracellular proteins such as protease and lipase. Also, Yarrowia lipolytica has been considered as an excellent host system for producing therapeutic glycoproteins, since it exhibits higher protein secretion efficiency than the traditional yeast Saccharomyces, has co-translational protein modification similar in animal cells (Boisrame et al., J. Biol. Chem., 273, p.30903-30908, 1998), has a lower number of mannose attached to the core chain than Saccharomyces cerevisiae (Madzak et al., J. Biotechnol., 10, p.63-81, 2004), and has no immunogenic alpha 1,3-linked mannose. To express and secrete therapeutic glycoproteins derived from human in Yarrowia lipolytica, the glycosylation pathway of Yarrowia lipolytica has to be understood, but is still poorly understood (Jaagar et al., Yeast, 20, p.633-644, 2003, Barnay-Verdier et al., Microbiology, 150, p.2185-2195, 2004).
To develop Yarrowia lipolytica as a host for secretory expression of therapeutic glycoproteins, the present inventors have conducted studies on the glycosylation pathway of Yarrowia lipolytica, and manufactured a strain comprising a disrupted YlOCH1 gene which mediates outer chain initiation. However, upon disruption of the YlOCH1 gene, the mannosylphosphorylation was found to be more activated.